Dataset Information

Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis.

ABSTRACT: BACKGROUND:Bordetella pertussis, the causative agent of whooping cough, is a highly clonal pathogen of the respiratory tract. Its lack of genetic diversity, relative to many bacterial pathogens, could limit its ability to adapt to a hostile and changing host environment. This limitation might be overcome by phase variation, as observed for other mucosal pathogens. One of the most common mechanisms of phase variation is reversible expansion or contraction of homopolymeric tracts (HPTs). RESULTS:The genomes of B. pertussis and the two closely related species, B. bronchiseptica and B. parapertussis, were screened for homopolymeric tracts longer than expected on the basis of chance, given their nucleotide compositions. Sixty-nine such HPTs were found in total among the three genomes, 74% of which were polymorphic among the three species. Nine HPTs were genotyped in a collection of 90 geographically and temporally diverse B. pertussis strains using the polymerase chain reaction/ligase detection reaction (PCR/LDR) assay. Six HPTs were polymorphic in this collection of B. pertussis strains. Of note, one of these polymorphic HPTs was found in the fimX promoter, where a single base insertion variant was present in seven strains, all of which were isolated prior to introduction of the pertussis vaccine. Transcript abundance of fimX was found to be 3.8-fold lower in strains carrying the longer allele. HPTs in three other genes, tcfA, bapC, and BP3651, varied widely in composition across the strain collection and displayed allelic polymorphism within single cultures. CONCLUSION:Allelic polymorphism at homopolymeric tracts is common within the B. pertussis genome. Phase variability may be an important mechanism in B. pertussis for evasion of the immune system and adaptation to different niches in the human host. High sensitivity and specificity make the PCR/LDR assay a powerful tool for investigating allelic variation at HPTs. Using this method, allelic diversity and phase variation were demonstrated at several B. pertussis loci.

SUBMITTER: Gogol EB

PROVIDER: S-EPMC1891110 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis.

Gogol Emily B EB Cummings Craig A CA Burns Ryan C RC Relman David A DA

BMC genomics 20070517

<h4>Background</h4>Bordetella pertussis, the causative agent of whooping cough, is a highly clonal pathogen of the respiratory tract. Its lack of genetic diversity, relative to many bacterial pathogens, could limit its ability to adapt to a hostile and changing host environment. This limitation might be overcome by phase variation, as observed for other mucosal pathogens. One of the most common mechanisms of phase variation is reversible expansion or contraction of homopolymeric tracts (HPTs).<h ...[more]

PMID: 17509142

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Project description:In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

Project description:Purpose: Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due at least in part to the introduction of a less effective acellular vaccine in the 1990’s. It is crucial then to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA/BvgS is the B. pertussis master regulator that regulates the expression of various virulence genes. Previous genome wide studies to identify the bvgAS regulon in B. pertussis have been limited to microarray analyses. The goal of this study is to define the bvgAS regulon using NGS based RNA-seq analysis and RT-qPCR and to compare the differences with the previous microarray data. Method: To define the bvgAS regulon, transcriptomes were generated using B. pertussis Tohama I BP536 (WT) and a ΔbvgAS derivative in the presence and absence of phosphorylated BvgA. Bacteria were grown either under modulating (50 mM MgSO4) conditions, in which phosphorylated BvgA is undetectable (Bvg- mode), or non-modulating (no MgSO4) conditions in which BvgA is phosphorylated (Bvg+ mode). After total RNA extraction and rRNA removal, strand-specific DNA libraries were prepared for Illumina sequencing using the ScriptSeq 2.0 kit (Illumina) in duplicates. Libraries were sequenced using a HiSeq 2000 sequencer (Illumina; University of Buffalo Next Generation Sequencing Core Facility). Sequence reads were mapped to the reference genome (NC_002929.2) and normalized against total reads. Differential expression analyses were performed using CLC Biomedical Genomic Workbench version 3.5.4 with default parameters. Differential gene expression profiles were obtained after Empirical DGE statistical test with FDR p-value. RT–qPCR validation was performed using SYBR Green assays. Result: Using CLC Biomedical Genomic Workbench version 3.5.4, ~ 20-25 million reads per sample were mapped to the reference B. pertussis genome (NC_002929.2). RNA-seq analyses identified >550 genes (15% of genome) that were regulated in a bvgAS-dependent manner with a fold difference ≥ 1.6 and FDR p-value < 0.05. RT-qPCR was used for confirmatory analyses for 80 genes. Overall, we identified 245 genes that were positively regulated and 326 genes that were negatively regulated by bvgAS. 362 members of the bvgAS regulon were newly identified by our study. Most importantly, our analyses indicated that the expression of dozens of transcriptional regulators increases, while the expression of multiple metabolic genes decreases in the presence of BvgA~P. Conclusion: We report here the first RNA-seq analysis of the bvgAS regulon in B. pertussis, revealing that > 550 genes are regulated by bvgAS. Our results suggest that metabolic changes in the Bvg- mode (absence of BvgA phosphorylation) may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new genes negatively regulated by bvgAS that can be tested for function.

Dataset Information

Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis.

Publications

Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis.

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